1. Field of the Invention
This invention relates to genetic modification of microbes to enhance production of a commercially important enzyme, beta-glucosidase. This invention also relates to genetic constructs that dramatically increase the amount of beta-glucosidase produced by microbes containing these constructs.
2. Background of the Related Art
The possibility of producing ethanol from cellulose has received much attention due to the availability of large amounts of feedstock, the desirability of avoiding burning or land filling the materials, and the cleanliness of the ethanol fuel. The advantages of such a process for society are described in the cover story of the Atlantic Monthly, April 1996.
The natural cellulosic feedstocks for such a process are referred to as "biomass". Many types of biomass, including wood, agricultural residues, herbaceous crops, and municipal solid wastes, have been considered as feedstocks for ethanol production. These materials primarily consist of cellulose, hemicellulose, and lignin. This invention can be applied to the conversion of the cellulose to ethanol.
Cellulose is a polymer of the simple sugar glucose connected by beta 1,4 linkages. Cellulose is very resistant to degradation or depolymerization by acid, enzymes, or micro-organisms. Once the cellulose is converted to glucose, the resulting sugar is easily fermented to ethanol using yeast. The difficult challenge of the process is to convert the cellulose to glucose.
The oldest methods studied to convert cellulose to glucose are based on acid hydrolysis (review by Grethlein, "Chemical breakdown of Cellulosic Materials", J.Appl.Chem. Biotechnol. 28:296-308 (1978). This process can involve the use of concentrated or dilute acids. The concentrated acid process produces a high yield of glucose, but the recovery of the acid, the specialized materials of construction required, and the need to minimize water in the system are serious disadvantages of this process. The dilute acid process uses low levels of acid to overcome the need for chemical recovery. However, the maximum glucose yield is only about 55% of the cellulose, and a high degree of production of degradation products can inhibit the fermentation to ethanol by yeast. These problems have prevented the acid hydrolysis process from reaching commercialization.
To overcome the problems of the acid hydrolysis process, cellulose conversion processes have focused more recently on enzymatic hydrolysis, using cellulase enzymes. Enzymatic hydrolysis of cellulose is carried out by mixing the substrate and water to achieve a slurry of 5% to 12% cellulose and adding 5 to 50 International Units (IU) cellulase enzymes per gm cellulose. Typically, the hydrolysis is run for 12 to 150 hours at 35-60.degree. C., pH 4-6.
Many microbes make enzymes that hydrolyze cellulose, including the wood rotting fungus Trichoderma, the compost bacteria Thermomonospora, Bacillus, and Cellulomonas; Streptomyces; and the fungi Humicola, Aspergillus and Fusarium. The enzymes made by these microbes are mixtures of proteins with three types of actions useful in the conversion of cellulose to glucose: endoglucanases (EG), cellobiohydrolases (CBH), and beta-glucosidase. EG and CBH enzymes are collectively referred to as "cellulose."
EG enzymes cut the cellulose polymer at random locations, opening it up to attack by CBH enzymes. As an example, Trichoderma strains produce at least four distinct EG enzymes, known as EGI, EGII, EGIII, and EGV.
CBH enzymes sequentially release molecules of cellobiose from the ends of the cellulose polymer. Cellobiose is the water-soluble beta-1,4-linked dimer of glucose. There are two primary CBH enzymes made by Trichoderma, CBHI and CBHII.
Beta-glucosidase enzymes hydrolyze cellobiose to glucose. Trichoderma makes one beta-glucosidase enzyme.
This final step in the cellulose hydrolysis which is catalyzed by beta-glucosidase is important, because glucose is readily fermented to ethanol by a variety of yeasts while cellobiose is not. Any cellobiose remaining at the end of the hydrolysis represents a loss of yield of ethanol. More importantly, cellobiose is an extremely potent inhibitor of the CBH and EG enzymes. Cellobiose decreases the rate of hydrolysis of the Trichoderma CBH and EG enzymes by 50% at a concentration of only 3.3 g/L. The decrease in rate of hydrolysis necessitates the addition of higher levels of cellulase enzymes, which adversely impacts the overall process economics. Therefore, the accumulation of cellobiose during hydrolysis is extremely undesirable for ethanol production.
Cellobiose accumulation has been a major problem in enzymatic hydrolysis because Trichoderma and the other cellulase-producing microbes make very little beta-glucosidase. Less than 1% of the total protein made by Trichoderma is beta-glucosidase. The low amount of beta-glucosidase results in a shortage of capacity to hydrolyze the cellobiose to glucose and an accumulation of 10 to 20 g/L of cellobiose during hydrolysis. This high level of cellobiose increases the amount of cellulase required by 10-fold over that if an adequate amount of beta-glucosidase were present.
Several approaches have been proposed to overcome the shortage of beta-glucosidase in cellulase enzymes.
One approach has been to produce beta-glucosidase using microbes that produce little cellulase, and add this beta-glucosidase exogenously to cellulase enzyme to enhance the hydrolysis. The most successful of such beta-glucosidase producing microbes have been Aspergillus niger and Aspergillus phoenicis. Beta-glucosidase from these microbes are available commercially as Novozym 188 from Novo Nordisk. However, the quantities required are much too costly for a commercial biomass to ethanol operation.
A second approach to overcoming the shortage of beta-glucosidase is to carry out cellulose hydrolysis simultaneously with fermentation of the glucose by yeast, the so-called simultaneous saccharification and fermentation (SSF) process. In an SSF system, the fermentation of the glucose removes it from solution. Glucose is a potent inhibitor of beta-glucosidase, so SSF is an attempt to increase the efficiency of beta-glucosidase. However, SSF systems are not yet commercially viable because the operating temperature for yeast of 28.degree. C. is too low for the 50.degree. C. conditions required by cellulase; operation at a compromise temperature of 37.degree. C. is inefficient and prone to microbial contamination.
A third approach to overcoming the shortage of beta-glucosidase is to use genetic engineering to overexpress the enzyme and increase its production by Trichoderma. This approach was taken by Barnett, Berka, and Fowler, in "Cloning and Amplification of the Gene Encoding an Extracellular .beta.-glucosidase from Trichoderma reesei: Evidence for Improved Rates of Saccharification of Cellulosic Substrates," Bio/Technology, Volume 9, June 1991, p. 562-567, herein referred to as "Barnett, et al."; and Fowler, Barnett, and Shoemaker in WO 92/10581, "Improved Saccharification of Cellulose by Cloning and Amplification of the .beta.-glucosidase gene of Trichoderma reesei," herein referred to as "Fowler, et al."
Both Barnett, et al. and Fowler, et al. describe the insertion of multiple copies of the beta-glucosidase gene into Trichoderma reesei strain P40. Both groups constructed plasmid pSAS.beta.-glu, a transformation vector containing the genomic T. reesei beta-glucosidase gene and the amdS selectable marker. The amdS gene is from Aspergillus nidulans and codes for the enzyme acetamidase, which allows transformed cells to grow on acetamide as a sole source of nitrogen. T. reesei does not contain a functional equivalent to the amdS gene and is therefore unable to utilize acetamide as a nitrogen source. The transformed cells contained 10 to 15 copies of the beta-glucosidase gene and produced 5.5-fold more beta-glucosidase than the untransformed cells.
The enhanced production of beta-glucosidase obtained by Barnett, et al. and Fowler, et al. is not sufficient to alleviate the shortage of beta-glucosidase for cellulose hydrolysis. The amount of beta-glucosidase made by natural Trichoderma strains, for example, must be increased at least 10-fold to meet the requirements of cellulose hydrolysis.
When overexpressing proteins in Trichoderma, one strategy is to link the gene of interest directly to the cbh1 promoter or to the cbh1 secretion signal. Since CBH1 is the most abundant protein produced by Trichoderma under cellulase-inducing conditions, the cbh1 promoter and secretion signal are thought to be very effective in directing the transcription and secretion of proteins encoded by a gene positioned after them in a genetic construct. Such a strategy has been successfully used to overexpress proteins from Trichoderma and other microorganisms (Margolles-Clark, Hayes, Harman and Penttila, 1996, "Improved Production of Trichoderma harzianum endochitinase by expression in Trichoderma reesei", Appl. Environ. Microbiol. 62(6): 2145-2151; Joutsjouki, Torkkeli and Nevalainen, 1993, "Transformation of Trichoderma reesei with the Hormoconis resinae glucoamylase P (gamP) gene: production of a heterologous glucoamylase by Trichoderma reesei", Curr. Genet. 24: 223-228; Karhunen, Mantyla, Nevalainen and Suominen, 1993,"High frequency one-step gene replacement in Trichoderma reesei 1. Endoglucanase I overproduction", Mol. Gen. Genet. 241: 515-522).
In spite of a significant amount of research effort, there has not been a means to produce sufficiently high levels of beta-glucosidase. Such a process would be a large step forward in the production of fuel alcohol from cellulose.